The adult, mammalian central nervous system (CNS) does not regenerate after injury despite the fact that there are many molecules present which promote nerve and axonal growth. The adult, mammalian peripheral nervous system (PNS), in contrast, does regenerate to some extent. It is believed that the lack of regeneration in the CNS is caused by the presence of molecules which actively prevent or inhibit regeneration. In the PNS, to the extent that neurons can regenerate, these inhibitors are thought to be removed or inactive and are thus not encountered by the re-growing axon. Hence, the well documented inability of the adult mammalian CNS to regenerate after injury is believed to result from a predominance of inhibitory molecules. There are at least three factors that are responsible for the lack of regeneration: the formation of a glial scar, inhibitors of regeneration in myelin and the intrinsic growth capacity of adult axons. The glial scar takes some time after injury to form. Therefore, it would be advantageous to encourage growth in this “window-of-opportunity”, before the scar forms. The main obstacles immediately after injury, therefore, are inhibitors of neuronal regeneration present in myelin.
To overcome these inhibitors, they could either be neutralized or the growth capacity of the axon could be changed such that the axons no longer respond to myelin by being inhibited. In this way, they would resemble young axons which regenerate in vivo and which are not inhibited by myelin in vitro. Previously, we showed that if the endogenous levels of cAMP are elevated in older neurons, either artificially with dibutyryl cAMP or by pre-treating the neurons with neurotrophins (“priming”), they are not inhibited by either myelin in general or by a specific myelin inhibitor, myelin-associated glycoprotein (MAG). See Cai, D. et al., Neuron 22:89-101 (1999); see also U.S. Pat. Nos. 5,932,542 and 6,203,792, the entire disclosures of which are incorporated herein by reference. We have also shown that the endogenous level of cAMP in young neurons is very high and that their ability to regenerate in vivo and to grow on myelin is cAMP-dependent (Cai et al., supra; 2001). The drop in neuronal cAMP concentration seems to parallel the developmentally regulated switch that decreases the ability of axons to regenerate. Thus, cAMP levels may play an important role in regulating the capacity of a neuron to undergo axonal regeneration. Downstream effectors which are directly responsible for improved neuronal growth on myelin remain unknown. One candidate may be polyamines, such as spermidine and spermine, and their diamine precursor putrescine, which are ubiquitously distributed in prokaryotic and eukaryotic cells and eukaryotic tissues.
Polyamines are involved in a large number of cellular functions, including many which involve nucleic acids, such as DNA replication, gene expression and peptide synthesis. (For a review, see, e.g., Tabor and Tabor, Annu. Rev. Biochem. 53, pp. 749-790 (1984)). Polyamines have been shown in some systems to be essential for cell proliferation and differentiation during wound healing. We considered it possible that, because polyamines are important in tissue wound healing, they may also function in axonal regeneration in the nervous system, which can occur with or without concomitant cell proliferation.
Polyamines are abundant in the mammalian nervous system (see, e.g., Bernstein et al., Prog. Neurobiol. 57(5), pp. 485-505 (1999)). And, polyamines have been shown to promote neurite regeneration of injured axons in cultured rat hippocampal neurons (Chu et al., Brain Res., 673, pp. 233-241 (1995)). Exogenous polyamine treatment has been reported to accelerate axonal regeneration and functional recovery of crushed peripheral (rat sciatic and facial) nerves (Dornay et al., Exp. Neurol., 92, pp. 665-674 (1986); Kauppila et al., Exp. Neurol., 99, pp. 50-58 (1988), Brain Res., 5775, pp. 299-303 (1992)). In the same peripheral nerve types, however, others have reported that exogenous polyamines had no significant effects on neural regeneration (Wong and Mattox, Exp. Neurol. 111, pp. 263-266 (1991)). Polyamines appear to enhance survival of certain sympathetic neurons after axonal injury (Gilad and Gilad, Brain Res., 724, pp. 141-144 (1988)). Polyamines also appear to play a role in neonatal nervous tissue development (Slotkin and Bartolome, Brain Res. Bull. 17, pp. 307-320 (1986)). Interestingly, biosynthesis of polyamines is enhanced in neurons treated with cAMP (Morris and Slotkin, J. Pharmacol. Exp. Therapeutic. 233, pp. 141-147 (1985)) or nerve growth factor (MacDonnell et al, Proc. Natl. Acad. Sci. U.S.A., 74, pp. 4681-4684 (1977)). Polyamines are therefore candidates for molecules which act in a cAMP-mediated signaling pathway to regulate neural regenerative capacity in the presence of myelin inhibitors.
Polyamines in mammalian cells are synthesized from the amino acid arginine in a pathway involving at least three catalytic steps. (See FIG. 1 for a schematic diagram of polyamine synthesis). The first step in polyamine synthesis is conversion of arginine to ornithine and urea, catalyzed by the enzyme arginase. Ornithine is converted to putrescine (a diamine) by the enzyme ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine synthesis (Shantz, L. M. and Pegg, A. E., Int. J. Biochem. Cell Biol.; 31(1), pp. 107-122 (1999); Wu, G. and Morris, S. M., Jr., Biochem. J. 336, pp. 1-17 (1998)). Putrescine, in turn, is the precursor to the polyamine spermidine, which can be converted to a variety of other polyamines, such as spermine (see FIG. 1).
In a number of cellular systems, ornithine decarboxylase (ODC) and arginase are co-induced (Salimuddin et al., Am. J. Physiol. 277, pp. E110-117 (1999)). Thus, arginase may regulate the availability of ornithine for polyamine synthesis despite the observation that conversion of ornithine to putrescine by ODC is the rate-limiting step in polyamine synthesis. Further support for this notion comes from observations that certain cells deficient in arginase cannot proliferate unless polyamines or ornithine are provided. For example, arginase activity is severely deficient in endothelial cells of the diabetic BB rat (an animal model of human type I diabetes mellitus) (Wu and Meninger, Am. J. Physiol. Heart Circ. Physiol. 265, H1965-H1971 (1993)), and these cells exhibit a marked impairment in proliferation (Meninger et al., J. Vasc. Research 33 (S1): 66 (1996)). Correlations between arginase activity and polyamine synthesis also been observed in kidney and intestine. A causal relationship between arginase activity and polyamine synthesis has not, however, been established.
Arginase genes are found in bacteria, fungi, plants and animals. Two isoforms of arginase encoded by separate genes have been identified in mammalian cells (Shi et al., Mammalian Genome 9, pp. 822-824 (1998); incorporated herein by reference). The two isoforms of arginase differ in molecular and immunological properties, tissue distribution, subcellular location, and regulation of expression. Type I arginase (arginase I) is a cytosolic enzyme which is highly expressed in liver and detected in only a few other tissues. Its main, but not its only, role is as a component of the urea cycle. Type II arginase (arginase II) is a mitochondrial enzyme which is expressed to varying degrees in a number of tissues but with little or no expression detectable in liver. One of the functions of arginase II is to regulate nitric oxide (NO) levels by competing with nitric oxide synthetase for arginine, the availability of which is one of the rate-limiting factors in cellular NO production (Gotoh and Mori, J. Cell Biol. 144, pp. 427-434 (1999)). NO, in turn, is important in the nervous system as a signaling molecule involved in cell survival, memory, and cell differentiation.
Arginase I and II are approximately 70% identical at the amino acid sequence level and differ primarily in that arginase II has a mitochondrial protein targeting sequence. (See, e.g., Morris et al., Gene 193, pp. 157-161 (1997); incorporated herein by reference). A comparison of arginase sequences from the livers of rat, human, Xenopus laevis, yeast and Agrobacterium TiC58 plasmid has revealed three conserved histidine residues. At the enzymatic level, the activities of arginase I and arginase II are similar but distinguishable because arginase II is not as susceptible to feedback inhibition by ornithine as is arginase I. There is reason to believe that arginase II, like arginase I, can increase cytosolic polyamines by increasing mitochondrial ornithine, which is then transported back to the cytosol. (See Jenkinson et al., Comparative properties of arginases, Comp. Biochem. Physiol., 114B, pp. 107-132 (1996)).
Arginase I and II are induced in murine macrophage cell lines by cAMP. A combination of cAMP, dexamethasone and lipopolysaccharides (LPS) leads to up-regulation of arginase I in kidney, but not in the small intestine, suggesting tissue specific regulation of arginase expression. It appears that arginase isoforms may play distinct but overlapping functional roles. To date, there is no data on the role of arginase isoforms in neuronal growth or axonal regeneration.
Recent data confirm the presence of both arginase I and arginase II in the central nervous system. In situ hybridization studies demonstrated that arginase II is distributed uniformly in the CNS in most neurons and astrocytes (Braissant et al., Brain Res. Mol. Brain Res. 70, pp. 231-241 (1999)). Immunohistochemical studies have demonstrated diffuse expression of arginase I protein in the CNS (Nakamura et al., Brain Res. 530, pp. 108-112 (1990)). Arginase II protein localization data is not available.
It would be useful to be able to regulate the inhibitors of axonal regeneration in neurons for treating patients with nervous system injuries or degenerative disorders where neural regeneration is a problem. As the molecular pathways that mediate neuronal growth and regeneration have not been precisely identified, it is difficult to design effective strategies to block the action of neuronal inhibitory molecules that prevent neural regeneration. In particular, it would be useful to be able to induce or otherwise increase arginase activity in neuronal cells to test whether such induction can—alone or in combination with other molecules—relieve inhibition of axonal outgrowth by myelin and myelin inhibitors, such as myelin-associated glycoprotein (NAG).